Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder in which exposure to volatile anesthetics or depolarizing muscle relaxants during surgery triggers uncontrolled Ca2+ release through sarcoplasmic reticulum (SR) ryanodine receptor (RYR) channels in skeletal muscle. The long term goal of this research is to define the molecular mechanisms that control RYR channels in situ, and to determine how anesthetics disrupt these mechanisms in the MH-susceptible (MHS) patient. Towards this goal, the proposed studies focus on identifying how agents that trigger or suppress the MH response affect the molecular interactions and structural events that underlie the gating of both MHS and normal RYR channels in situ. These studies will utilize the pig RYR1 Arg615Cys model of MH to address three specific aims. Aim I will determine the role of calmodulin (CaM) as a physiologic effector of MHS and normal RYR1 channels. [125I]CaM binding properties of the different channel isoforms (RYR1, RYR2, and RYR3) will be characterized, and compared with CaM's functional effects on the isolated channels as determined using [3H]ryanodine binding, Ca2+ flux, and single channel measurements. Effects of the Arg615Cys mutation on RYR1 [125I]CaM binding will also be characterized to determine the basis of the increased CaM-dependent activation of MHS channels. Related experiments will define CaM's in situ role controlling SR Ca2+ release in permeabilized fiber preparations, where the architecture of excitation-contraction (E-C) coupling remains intact. Aim II will define the mechanism of action of dantrolene, the specific treatment of MH. Experiments using recombinant, heterologously expressed RYRs will test the hypothesis that dantrolene inhibition of SR Ca release reflects a direct action of this agent at the RYR1. Characterization of dantrolene's functional effects on RYR1 in isolated preparations will be complemented with investigations of dantrolene's effects on SR CA2+ release in muscle fiber preparations. Aim III will identify the structural events that underlie gating of MHS and normal RYR1 channels. Fluorescently-labeled FKBP12 and CaM bound to specific regions on the RYR1 will reveal specific actions of the MHS mutation, dantrolene, and anesthetics on rotational and structural transitions of the RYR1 protein. MH remains a significant cuase of anesthetic-induced death and is an important model for a variety of disorders characterized by a loss of intracellular Ca2+ homeostasis. Identification of the molecular and biophysical mechanisms that underlie the triggering and suppression of the MH response will aid in the development of improved strategies for the prevention and treatment of this life-threatening response to anesthetics.